Conjugative transfer of genetic traits is mediated by a wide range of plasmids and transposons, and can occur between species and even kingdoms. Although first described over 50 years ago, we still have only a rudimentary knowledge of the molecular details surrounding this important mechanism for DNA transfer. Detailed knowledge of the conjugative mechanism is, therefore, of critical importance. The long range goal of this project is to understand, at a molecular level, the mechanistic details of conjugative DNA transfer. Previous studies indicate that DNA transfer begins at a site- and strand-specific nick (nic) in the conjugative plasmid, which is then unwound as ssDNA is transferred into the recipient. This laboratory has shown, using the F plasmid as a model, a requirement for two F-encoded proteins, Traip and TraYp, and one host-encoded protein, integration host factor (IHF), in the nicking reaction; subsequent unwinding has not yet been reconstituted in any system. Four specific aims are proposed. The 1st aim will focus on the role of TraYp and IHF in the Tralp-catalyzed transesterification reaction. Preliminary data suggest IHF may bind to one of two mutually exclusive sites that provide a molecular switch for initiating conjugation that is either on or off. This will be explored using chemical footprinting and IHF binding site mutants. In addition, TraYp + tHE may alter the DNA structure surrounding nic such that the DNA has ssDNA (or non-B DNA) character. The 2nd aim is to reconstitute the coupled nicking-unwinding reaction catalyzed by Tralp. Initial studies indicate a previously unrecognized host protein is required to "trigger" unwinding of DNA nicked by Tralp. This protein will be purified, using a biochemical complementation assay, and characterized in terms of its interaction with Tralp and its role in strand transfer. The F plasmid model provides the best possibility of reconstituting this key reaction because the helicase and site-specific nicking activities have been identified and a minimal relaxosome has been reconstituted. The 3 about aim will define the catalytic residue(s) in Traip involved in the site- and strand-specific transesterification reaction. Preliminary results indicate the involvement of two tyrosines, Y16 and Y23. The role of each tyrosine will be evaluated by constructing specific mutants and evaluating each mutant in vitro and in vivo. We also propose to crystallize the Tralp transesterase domain in the presence and absence of an oligonucleotide substrate to gain insight into the interaction of the transesterase with its substrate. The final aim will focus on the role of TraMp in the initiation reaction. Genetic studies indicate a role for this protein but biochemical details are lacking. The protein will be purified and used in relaxosome reconstitution studies. Taken together, the results gained from these experiments will advance our understanding of the mechanism of conjugative DNA transfer and will pave the way for future experiments to look at transfer across the cell membrane.